Plant lipids such as seedoil triaclyglycerols (TAGs) have many uses, for example, culinary uses (shortening, texture, flavor), industrial uses (in soaps, candles, perfumes, cosmetics, suitable as drying agents, insulators, lubricants) and provide nutritional value. There is also growing interest in using plant lipids for the production of biofuel.
Biofuel
Growing demand for alternative sources of energy can be fulfilled at least in part with a renewable supply of plant-derived biofuel. To be a viable alternative to fossil fuels, the biofuel should provide a net energy gain in production, have environmental benefits, be economically competitive, and producible in large quantities without reducing food supplies, a current unintended byproduct of existing biofuel production.
Plants represent a significant source of lipids because many species accumulate lipids as major storage components in seeds. The main form of vegetative storage lipids in seeds, which represent, depending on the species, 15-50% of seed weight, is triacylglycerol (TAG). However, the primary substrate for lipid synthesis are the carbohydrates generated in green photosynthetic tissues (leaves and stems) that are subsequently metabolized in chloroplasts to produce free fatty acids and acetyl-coenzyme A (acetyl-CoA) units, the basic building blocks for TAG. Therefore, plant leaves are the main place of building block synthesis for TAG. The amount of TAG accumulated in oilseeds may be in part, determined by the amount of fatty acid produced in plastids (Bao and Ohlrogge, 1999). Final storage of TAG occurs in seeds in small spherical organelles termed oil bodies. Only about 0.2-0.3% of leaf biomass is represented by TAG.
High biomass plants, particularly broad leaf high biomass plants, have great biofuel potential. Plants that can yield between 100-400 tons/acre of low-cost, high-value biomass materials are particularly useful, especially when there is none of the high costs, labor requirements, chemical inputs, or geographic restrictions associated with low biomass plant production.
Monoacylglycerol Acyltransferases
The monoacylglycerol acyltransferase (MGAT) enzyme is associated with mammals, primarily with the intestine in mammals where it catalyzes the synthesis of diacylglycerol (DAG) directly from monoacylglycerol (MAG) and fatty acyl-CoA. In contrast, the primary TAG synthesis pathway found in plants is the Kennedy, or glycerol phosphate pathway (FIG. 1) which does not include a MGAT step. In the Kennedy pathway, DAG is formed from an acylated glycerol backbone in a two-step reaction consisting of an initial acylation by lysophosphatidic acid acyltransferase (LPAAT) which adds a fatty acyl-CoA to a lysophosphatidic acid (LysoPA; LPA) substrate and the subsequent removal of a phosphate group from the product, phosphatidic acid (PA), to yield inorganic phosphate (Pi) and DAG. In contrast, MGAT catalyzes the formation of DAG directly, by acylating a MAG with an acyl group coming from fatty acyl-CoA. Following synthesis of DAG, another enzyme, diacylglycerol acyltransferase (DGAT), acylates DAG to form TAG.
The first MGAT gene to be isolated was from mouse (MGAT1) and this gene coded for a membrane-bound, non-soluble, enzyme (Yen et al., 2002). Other similar MGAT genes have been characterized in animals, including a second MGAT gene from mouse (MGAT2) and three human genes, but no genes encoding MGAT have been confirmed to have been cloned from plants (Cao et al., 2003; Cheng et al., 2003).
Diacylglycerol Acyltransferases
DGAT is an integral membrane protein that catalyzes the final enzymatic step in the production of TAG in plants, fungi and mammals. This enzyme is responsible for transferring an acyl group from acyl-coenzyme A (acyl-CoA) to DAG to form TAG. DGAT is associated with membrane and lipid body fractions in plants and fungi, particularly, in oilseeds where it contributes to the storage of carbon used as energy reserves. DGAT is known to regulate TAG structure and direct TAG synthesis. Furthermore, it is known that the DGAT reaction is specific for lipid synthesis. Overexpression of the acyl-CoA-dependent DGAT in a seed-specific manner in wild type plants results in augmentation of seedoil deposition and average seed weight (Jako et al., 2001).
To maximise yields for the commercial production of lipids, there is a need for further means to increase the levels of lipids, particularly non-polar lipids such as DAGs and TAGs, in transgenic organisms or parts thereof such as plants, seeds, leaves, algae and fungi.